A cGMP-stimulated cyclic nucleotide phosphodiesterase has been purified more than 13,000-fold to apparent homogeneity from calf liver. The purified enzyme exhibited a singleprotein-staining band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 102,000, a sedimentation coefficient of 6.9S, and Stokes' radius of 67 A. From these values an apparent molecular weight of 201,000 and frictional ratio (f/fo) of 1.7 were calculated. These hydrodynamic properties suggest that the native enzyme exists as a nonspherical dimer of similar, if not identical, polypeptide chains. The apparent Km's were 22 and 40 muM for cGMP and cAMP, respectively. The Vmax's and turnover numbers were 87 mumol/min/mg of protein, 17,500/min for cGMP and 79 mumol/min/mg of protein, 15,800/min for cAMP, respectively. The hydrolysis of both cyclic nucleotides exhibited positive homotropic cooperativity with Hill coefficients of 1.8 for cAMP and 1.2 for cGMP. The rate of cAMP hydrolysis by the purified phosphodiesterase when measured at 0.5 muM cAMP was enhanced more than 30-fold by cGMP with an apparent activation constant (Ka) of 0.35 muM.